Field of the Invention
The invention relates, in general, to apparatus and methods for performing assay on biological tissue.
In situ assays are performed on tissues by using reagents to detect macromolecules directly in cells. Standard in situ assays include in situ hybridization (ISH) and immunocytochemistry (IC). Such assays utilize suitable probe reagents, including nucleic acids and/or antibodies, to bind with and mark message RNA or protein in cells to detect the presence, location and quantity of such molecules. Such methods, particularly those that are also capable of detecting low abundance RNA in relatively small cell samples, are of great importance in clinical oncology for early detection, diagnosis, prognosis and treatment of cancers.
In a typical in situ assay, a specific amount of a reagent is added to a tissue section mounted on a slide. The reagent is allowed to bind to the tissue for a predetermined amount of time, i.e., one to several hours, so as to mark specifically bound message RNA or protein in the tissue section. The tissue section is then washed to remove non-specifically bound reagent from the tissue and is further processed to prepare tissue for analysis via microscopic examination, etc.
In situ methods currently employed use an "open-bench" technique in which the reagent is added manually to a tissue section mounted on a slide and then each slide is individually washed. Long incubation times required for ISH reactions usually require that reactions are carried out under a thin film or cover sheet placed over the tissue section on the slide which is then sealed with wax or oil to prevent drying high temperature incubation. These methods are clearly very labor intensive and time consuming and prone to ad hoc variations which reflect high cost and poor reliability.
Recently, more automated assay apparatus and methods have been devised to address and overcome certain of these problems. Such automated apparatus utilize a slide or cover plate that fits on a tissue carrying slide with a narrow (approximately 0.2 mm) clearance between the two slides. Fluid is introduced to the tissue section by capillary movement between the two opposed slides and surface adhesion holds the liquid reactant in place between the two slides to create an open reaction chamber. However, while such devices are a major advance in performing in situ assays, they have significant defects which limits their reliable use. These automated units use capillary action and surface adhesion which limits their versatility to certain reactions and create sensitivity problems. For example, such automated apparatus cannot be used at all for ISH assays in which a strong ionic detergent (i.e., SDS) is a preferred reagent and weak non-ionic surfactants (i.e., triton) have to be present at high (approximately 3.0 percent levels for efficient operation. Viscous reagents which give versatility to ISH assays are also problematic for these capillarity-based automated devices.
Further, the close tolerance between the two opposed slides in each unit creates an adhesive fluid surface which limits reactant mixing during reactions since the tissue occupies only a small fraction of the slide area and the reactants are spread over a larger slide area and, also, makes washing of unused reactant very inefficient. Evaporation is also a problem with the open chambers employed in these devices. Thus, these automated in situ assay devices are reliable for only a few applications and are primarily used for abundant targets where the signal to noise ratio is not a critical factor.
Thus, it would be desirable to provide an in situ apparatus and method which still utilizes a thin chamber for use of a small volume of reagents, but in which reactant fluid position is controlled by gravity on a tissue carrying slide rather than by capillary action. It would also be desirable to provide an in situ assay apparatus in which the reaction chamber has sufficient vertical space between a cover slide and the tissue carrying slide to reduce friction for complete reactant mixing. It would also be desirable to provide an in situ assay apparatus and method which makes washing of the hybridized tissue more thorough than previously devised in situ assay techniques. It would also be desirable to provide an in situ apparatus and method in which no evaporation of the reactant can occur and only a small volume of the reactant is used to cover only the tissue on the slide. It would also be desirable to provide an in situ apparatus and method which combines blocking and probe-incubation steps into a single unit. It would also be desirable to provide an in situ apparatus and method in which predetermined amounts of pre-prepared reactant can be mounted in place on the slides in advance of the in situ assay.